Composition and method for testing biological fluids



treatment of diabetes.

Patented Aug. 26; 1952 COMPOSITION AND METHOD FOR TESTING BIOLOGICAL FLUIDS Richard M. Carson and Reuben R. Sacks, Dayton, Ohio No Drawing. Application January 30, 1951,

I Serial No. 208,668 r 11 Claims. (01. 252-408) Thisinvention relates to a composition and method for testing biological fluids to determine the presence of reducing substances therein, and is designed primarily for determining the presence of and the amount of sugar in urine.

This application is a continuation in part of our copending application Serial No. 579,314, filed February 22, 1945,- for Composition and Method forTesting Biological Fluids, now abandoned. l l

In the diagnosis and treatment of certain diseases it is essential to ascertain whether the urine or blood contains sugar or alcohol and the amount thereof. This is particularly true in the No cure for that disease has been found butit can usually be controlled by the regulation of the diet or by the administration of insulin, or both. Thecharacter of thediet and/or the dosage of insulin are determined largely by the amount of sugar content in the urine, which maychange from time to time. It is desirabletherefore that the patient shouldbe able to test his urine periodically to determine what if any change in treatment is necessary, suchchange's of course being made under the instructions from his physician. There are various materials and methods by which the urine may be so tested and some of these have been made available to diabetic patients in the form of equipment, or kits, intended for individual use in the home. These tests have not been well adapted for'use in the home but have, for the'most part at least, involved one or more objectionable characteristics.

Usually, the presence of sugar isindicated by the. change in the color of the testing reagent and the amount of sugar is indicated by the particular shade of resulting color, and is detcrmined by comparison'with a color chart. Such a com-v rials required are usually expensive and in some j instances lack stability and. quickly deteriorate. One object of theinvention is to providesuch a test which can be easily and accuratelyeffected by the ordinary patient. 1

A further object of the invention is to provide such a test which can be effected without the application of external heat.

A further object of the invention is to provide such a test which does not require a comparison of colors. l

A further object of the invention is to provide such a test which is both qualitative and quantitative.

A further object of the invention is to provide such a test which can be effected quickly and at a low cost.

. A further object of the invention is to provide such atest in. which the result will not be materially affected byother reducing substances in thefluid being tested.

A'further objectof the invention is to providean improved reagent for effecting such a test; l

A further object of the invention is to provide such a reagent which will react with the reducin substances inthe fluid being tested to form a composition the color of which will be the same regardless of the quantity of the reducing substance present in the fluid.

A further object of the invention is to provide such a reagent the reaction of whichwith the reducing substance will be completed in a period of time determined by the amount of reducing substances present in the fluid being tested.

Other objects of the invention may appear as the reagent and method are described in detail.

In practising the invention we provide a testing medium or reagent in the form of a stabilized solution of a color indicator or color imparting agent which will react with a reducing agent such as sugar oralcohol to noticeably change color.

portion of reducing agent in the biological fluid, can be determined bythe length of time for the reaction rather than by color gradation. For: this purpose we have found that the desired re-' sults are obtained where we employ as the color indicator, a manganate of an alkali metal such as lithium, sodium, potassium, rubidium and cesium. Potassium or sodium manganate ora mixture thereof are preferably employed because they are more readily available at an economical cost.

The alkali-metal manganate isprepared in aqueous solution and to stabilize the manganate we employ a stabilizing agent. The stabilizing agent should be alkali, it should dissolve readily in water and should not react with the alkali metal manganate nor should it interfere with or inhibit the reaction between the alkali metal manganate and the reducing agent. We accomplish the desired results by employing the hydroxide of an alkali metal. We prefer to employ sodium or potassium hydroxide or a mixture thereof because these are more readily available at an economical cost.

As previously indicated, the alkali metal manganate and alkali metal hydroxide are dissolved in water to provide the desired test reagent. The proportions may be varied within limits. However, we have found that the proportion of alkali metal manganate to alkali metal hydroxide in the solution should be approxi- 'mately between 0.1% and 2.6% by weight. The

solution may be prepared in concentrated form and later diluted with more Water just prior to use or it may be prepared, packaged, shipped and stored for reasonable periods of time in the proportions for use. In either event, the aqueous solution or'reagent when ready for use should contain approximately between and 40% by Weight of alkali metal hydroxide and approximately between 0.04% and 04% by weight of alkali metal manganate.

The several components of the solution may be prepared in any desired manner and then dissolved in the water in the desired concentrations. However, we have found that the reagent may be conveniently produced by preparing a strong solution of an alkali metal permanganate in distilled water and combining with this solution an alkali metal hydroxide in. a quantity to produce a solution in which the proportion of alkali metal manganate will be between 0.1% and 2.6% by weight of the alkali metal hydroxide. The hydroxide and permanganate will react with the result that all of the permanganate will be converted to the manganate. Specific examples of reagents embodying our invention and the methodof preparing them are as follows:

Esrample No. 1

hydroxide (NaOI-l.).. This solution isstable and strongly oxidizing in character, is green in color and. will react at room temperatures on a reducing substance in the fluid being tested to change its color to areddish-brown. In prepar ing, the reagent accordance with the foregoing. example, permanganates of any of the, other alkali metals or mixtures thereof may be substituted for the potassium permanganateand hydroxides of any'of the other alkali metals or mixtures thereof may be substituted for the sodium hydroxide.

Example No. 2

0.05 grams of chemically pure potassium The, final solution comprises; a mixture of equal. molecular parts of sodium, mahganate (liazlVlnQa)v and potassium manga nate. (KzMnOi) in a. strong solution of sodium permanganate is dissolved in approximately 15 cubic centimeters of distilled water and 15 grams of sodium hydroxide is added to the solution and thoroughly mixed therewith. After the reaction is completed, additional distilled water is added thereto so as to increase the total volume of water to eighty-five cubic centimeters. The resulting solution is stable and strongly oxidizing in character, is green in color and will react at room temperatures on a reducing substance in the fluid being tested.

In the foregoing example the permanganate of any of the other alkali metals or mixtures thereof may be substituted for the potassium permanganate and the hydroxide of any of the other alkali metals or mixtures thereof may be substituted for the sodium hydroxide. Also, instead of adding the full amount of distilled water as indicated the solution may be left in more concentrated form and packaged, shipped and stored in this fashion to be diluted to the indicated amount immediately prior to use.

Example No. 3

0.2 grams of chemically pure sodium permanganate is dissolved in approximately 15 cubic centimeters of distilled water and 25 grams of sodium hydroxide is added to the solution and thoroughly mixed hterewith. After the reaction is completed, additional distilled water is added thereto so as to increase the total volume of water to seventy-five cubic centimeters. The. resulting solution is stable and stronglyoxidizing in character, is green in color and will react at room temperatures on a reducing substance in the fluid being tested.

In the foregoing example the permanganate of any of the other alkali metals or mixtures thereof may be substituted for the sodium permanganate and the hydroxide of any .of the other. alkali metals or mixtures thereofmay besubstituted for the sodium hydroxide. ,Also, instead of adding the full amount of distilled water as indicated the solution may be left in more con-'- centrated form and packaged, shipped and stored in this fashion to be diluted to the indicated amount immediately prior to use.

Example No. 4 a 0.3 grams of chemically pure potassium per manganate is dissolved in approximately 15 cubic centimeters of distilled water and 40 grams'of potassium hydroxide is addedto the solution and thoroughly mixed therewith. After the reaction is completed, additional distilled water is added thereto so as to increase the total volume of water to sixty cubic centimeters. The resulting solution i stable and strongly oxidizing in character, isgreen in color and will react at room temperatur'eson a reducing substance being tested. I

In thev foregoing example the permanganate of any. of the other alkali metals or mixturcstherein: the-fluid of; may be substituted for the potassium perman-- ganateand the hydroxide of any of the other,

in this fashion to be diluted to the indicated amount immediately prior to use. I

A reagent embodying our present invention and made in'accordance with any oftheforegoing examples is particularly useful in' determining the presence of and the relative amount of su'garlinurine but with. minor modiflcationslit may-also be used. for other similar tests such. as testing blood for. either sugar oralcohol or. testingrurine for alcohol. Themethod of testing mayrvary. but we prefer to perform the testin the manner hereinafter described. 1

The sugar. whichginjtheformpf glucose, is present vin varying; amounts in diabetic urine is a stronglyreducing substance which reacts with the oxidizing reagent. toproduce manganese. die- :oxide..(MnOz), which is a precipitate of reddish brown color and changes the color of the solution from the original green to the easily distinguishable.reddishbrown. In making the tests the proportions of urine and the reagent aredetermined in part by the strength of the reagent andin partby the speed with which thereactlonis tobe effected. While the-reaction is quickly completed it is not instantaneous-and the time interval varies with the amount of sugar in the urine, the timefrequired for completion decreasing as the amount of sugar increases. The reaction of the reagentwiththe sugar changes the color of the solution to the same reddish brown regardless of the amount of sugar pres! ent, and the amount of sugar present is indicated by the time required for the reaction to take ;place. v i

In .making the test a few drops of urine are placed iin.a. clean, dry, transparent receptacle of a suitable size and shape, preferably a test tube of one-half inch diameter. A predeterminedquantity of the reagent is ,then added to the urine, preferably all atone'time, and :the time required for the mixture to change from a green color to a reddish brown color is noted. The proportion of urine to reagent may vary in accordance with the character of the reagent or the desired speed of reaction. For instance, with a reagent having the concentration above set forth in Example N0. 1, it is preferable to place five drops of urine in the test tube and to add thereto one cubic centimeter of the reagent. It is customary to designate, or rate, the quantity of sugar present by numerals, usually followed by the word plus, such as 1 plus, 2 plus, 3 plus and 4 plus, which cover a range suitable for tests by the patient. When a complete change of color takes place in twenty seconds or less the rating is 4 plus. If a complete change of color requires more than two minute the amount of sugar is not of material importance and the result is considered negative. The time intervals corresponding to the several ratings are shown in the following table, which is furnished to the patient:

Quantity of Glucose Time Present 4 plus 2% or more. 3 plus 1%.

2 plus 0.75%.

1 plus 0.50%. Negative.

dize other. reducing substances containedin the urine and therefore such other. substances, do not in any way interfere with the, test. J i The foregoing test for determining thepresence and the quantity of sugar in urine contemplates using a reagent made in accordance with Example No. 1. It will be appreciated that reagents with lvarying proportions of alkali metal manganate and alkali metal hydroxide within the limits set forth above may also beemployed and that the time of. reaction will vary as the proportions of ingredients are varied. Thus, if the proportion of either the alkali metal hydroxide or the alkali metal manganate are increased Within the indicated proportions, the reaction time is increased and vice versa. The reaction times of reagents of varying formulae may be readily determined by1simple tests with a given quantity of urine having known percentages of sugar. It willalso be understood that ,the reaction-{time will. be changeduby, varying the relative amount of urine used in the tests. Thus, where arelatively larger quantity of urine isflemployed the reaction. time will be speeded up.-.... i From the;foregoing,jit will be appreciated that our test and our testing reagentgis very simple in character. Its technique is easily acquired. no knowledge or chemical reactions is required and it is not necessary tocompare various shades of colorswitha color chart. Therefore, itis well suited for use by patients. Furthermore, the test requires very little; equipment, no external heat and only one reagent. It is effected at room temperatures and produces no objectionable odors. Thus it is convenient for use. in privacy in the home or when. traveling; Moreover, the cost per test is low.

While we have described several examples of a preferred reagent and method of preparing and using the same we wish it to be understood that we do not wish it to be limited to the details thereof as various modifications may occur to persons skilled in the art.

We claim:

1. Th method of testing a biological fluid which comprises adding to the fluid a solution of sodium manganate in an alkali metal hydroxide to oxidize the reducing substance in the fluid.

2. The method of testing a biological fluid which comprises adding to the fluid a reagent containing sodium manganate, potassium manganate and sodium hydroxide to oxidize a reducing agent in said fluid. Y

3. The method of testing a biological fluid which comprises adding to the fluid a reagent containing sodium manganate, potassium manganate and potassium hydroxide to oxidize a reducing agent in said fluid.

4. The method of testing urine for sugar which comprises dissolving potassium permanganate in water, adding sodium hydroxide to said solution to form an oxidizing reagent, placing a predetermined quantity of urine in a test tube, adding to said urine a predetermined quantity of said reagent.

5. A method of testing a biological fluid which comprises adding a sample of the fluid to a reagent containing sodium manganate, potassium manganate and sodium hydroxide to eifect oxidization of the reducing substances in said fluid at a rate which will permit the time required for oxidization to be measured.

6 The method of testing a biological fluid which comprises mixing with the fluid an aqueous solution of approximately between 0.04% and aeoasee '0.4%- by weight ofnan alkali metal manganate and approximately between 15% and 40% by weight of an alkali metal hydroxide.

7. The method of testing a biological fluid which comprises mixing with the fluid an aqueous solution of approximately between 0.04% and 0.4% by weight of'a'manganate of a metalselected from the group consisting of sodium and potassium and approximately between 15% and 40% by weight of an hydroxide of a metal selected from the group consisting of sodium and potassium.

8.1 Areagent for determining the presence and proportions of reducing agents such as glucose in biological fluids by a marked change of color of the reagent within predetermined periods of time and without the application of external heat comprising an aqueous solution of. an alkali metal manganate and of an alkali metal hydroxide in which the alkali metal manganate comprises approximately between 0.1 and 2.6 by weight of the alkali metal hydroxide.

9. A reagent for determining the presence and proportion of reducing agents such as glucose in biological fluids by a marked change of color of the reagent within predetermined periods of time and without. the application of external heat comprising an aqueous solution of a man-ganate of a metal selected from the group consisting of sodium and potassium and an hydroxide of a metalselected from the group consisting of sodium and potassium in which the metallic manganate comprises approximately between 0.1% arid-2.'6% by weight of the metallic hydroxide.

it). A reagent for-determining the presence and proportion of reducing agents such as glucose in biologicalfluids by a marked change. of color .01 the reagent Within predetermined periods of time and without the application of ext'ernalheatcomprising an aqueous solution of between-approximately 0.04% and 0.4% by weight of alkali metal manganate and approximately between 15% and by weight of an alkali metal hydroxide.

ll. A reagent for determining the presenceand proportion of reducing agents such as glucose in biological fluids by a marked change of color of the reagent withinpredetermined periods .0! time and without the .applicationof external heat comprising an aqueous solution of approximately between 0.04% and 0.4% by weight of a manganate of a metal selected from the group consisting of sodium and potassium and approximately :between 15% and 40% by weight of an hydroxide of a metal selected from the group consisting of sodium and potassium.

RICHARD M. CARSON. IRETJBZEN R. SAEKS.

REFERENCES CITED The following references are of record in the OTHER REFERENCES Mellor, Comprehensive Treatise on Inorganic and .Iheoretical Chemistry, "1:932, vol. 12 Longmans Green 8: (10., :N. Y pps. 283, 284, 2,86 and 288. 

5. A METHOD OF TESTING A BIOLOGICAL FLUID WHICH COMPRISES ADDING A SAMPLE OF THE FLUID TO A REAGENT CONTAINING SODIUM MANGANATE, POTASSIUM MANGANATE AND SODIUM HYDROXIDE TO EFFECT OXIDIZATION OF THE REDUCING SUBSTANCES IN SAID FLUID AT A RATE WHICH WILL PERMIT THE TIME REQUIRED FOR OXIDIZATION TO BE MEASURED.
 8. A REAGENT FOR DETERMINING THE PRESENCE AND PROPORTIONS OF REDUCING AGENTS SUCH AS GLUCOSE IN BIOLOGICAL FLUIDS BY A MARKED CHANGE OF COLOR OF THE REAGENT WITHIN PREDETERMINED PERIODS OF TIME AND WITHOUT THE APPLICATION OF EXTERNAL HEAT COMPRISING AN AQUEOUS SOLUTION OF AN ALKALI METAL MANGANATE AND OF AN ALKALI METAL HYDROXIDE IN WHICH THE ALKALI METAL MANGANATE COMPRISES APPROXIMATELY BETWEEN 0.1% AND 2.6% BY WEIGHT OF THE ALKALI METAL HYDROXIDE. 